Finding out the blood coagulation time, designated as prothrombin time (PT), i.e. the aptitude of different proteolytic enzymes, also known as “factors”, to contribute to the formation of a clot, or conversely, to prevent it, forms part of routine examinations, or even daily examinations in numerous acquired, traumatic, pre or post-operative pathological situations. It is, for example, necessary, during anticoagulant treatment for heart disease, to be able to adjust the dosage of an anti-thrombotic medicine, for example warfarin or heparin, in order to prevent any risk of haemorrhagy in the event of an overdose, or conversely, the risk of thrombosis if the anticoagulant dose is insufficient.
This determination of prothrombin time (PT) or partially activated thromboplastin time (APPT) has long been carried out in a laboratory by direct visual observation of the time necessary for a clot to form, then with the help of more or less complex and cumbersome apparatus usually relying upon optical detection, such as those disclosed for example in U.S. Pat. Nos. 5,302,348 and 5,154,082.
According to most recent methods, the principle consists in using a chemical substrate incorporating at least one chemical reactant, an end link of which can be cut by a specific enzyme to release a group (LG) whose presence can be detected in the measuring medium by a signal representative of the enzyme activity.
This method of detection corresponds for example to that disclosed in EP Patent No. 0 679 193. In the method disclosed, a sensor includes a chemical substrate, an end link of which can be cut by the enzyme being analysed to release a group (LG) whose concentration representative of activity of the enzyme in the medium can be measured by optical means based on alterations in colorimetry, luminescence or fluorescence. When the bodily fluid being analysed is whole blood, the red blood cells have to be removed, either by prior centrifugation of the sample, or by providing a membrane forming a barrier to the red blood cells on the sensor. This method thus has the drawback of requiring a relatively long, even expensive analysis time, to remove the red blood cells.
The aforementioned drawback can be greatly reduced, even removed, with the method proposed in EP Patent No. 1 031 830 and in U.S. Pat. No. 6,352,630 B1, both of which are incorporated in this Application by reference. The method, which concerns the blood coagulation measurement time, also relies on the indirect determination of the activity of a proteolytic enzyme by means of a chemical substrate able to release, via the action of the enzyme, leaving groups which will alter the electric properties of the medium, the resulting signal being in this case analysed by amperometry and correlated with a PT or APTT value representative of the coagulation time. With this non-colorimetric method, the prior preparation to obtain clear plasma is omitted, and determination can be carried out more quickly on whole blood.
All of the methods that have just briefly been recalled only allow an overall determination to be carried out and do not identify, among all the enzymes involved in the coagulation phenomenon, the enzyme responsible for a coagulation anomaly, whether this be haemophilia or thrombosis.
Until recent times, in order to obtain this kind of information, the method consisted in separating a blood sample into several samples and causing reactions with various anti-bodies to identify which enzyme was defective. This method required a relatively large blood sample, necessitated a lot of time and could only be carried out in a laboratory.
More recently, International Patent Application No. WO 03/093831 discloses a method for determining in real time the evolution of thrombin activity in a blood sample, but preferably in a plasma sample, relying upon fluorometric determination, compared to a calibration curve. This method has the same drawbacks as those previously cited for overall prothrombin time determination, concerning in particular the relatively large volume of the sample (approximately 160 μl, 80 μl of which is for the calibration solution), and the rather long measuring time (approximately 45 minutes).